Vaccine based on membrane bound proteins and process for making them

ABSTRACT

Disclosed is an invention related to the preparation and use of vaccines against pathogenic organisms, such as herpes virus. The vaccines hereof are based upon the use of glycoproteins of the organism, that have been prepared via recombinant means, and preferably C-truncated versions thereof. These glycoproteins when incorporated into a vaccine composition afford protection against pathogenic challenge after administration.

This application is a continuation of application Ser. No. 08/171,858,filed Dec. 21, 1993, now abandoned which is a continuation ofapplication Ser. No. 07/814,243, filed Dec. 23, 1991, which is acontinuation of Ser. No.07/695,585, filed May 5, 1991, now abandoned,which is a continuation of application Ser. No. 06/878,087, filed Jun.24, 1986, now abandoned, which is a continuation of application Ser. No.06/588,170, filed Mar. 9, 1984, now abandoned, which is acontinuation-in-part of application Ser. No. 06/527,917, filed Aug. 30,1983, now abandoned and of application Ser. No. 06/547,551 filed Oct.31, 1983 both now abandoned.

This invention relates to membrane bound proteins and derivativesthereof, and to vaccines obtained from them.

BACKGROUND

Analysis of the immune response to a variety of infectious agents hasbeen limited by the fact that it has often proved difficult to culturepathogens in quantities sufficient to permit the isolation of importantcell surface antigens. The advent of molecular cloning has overcome someof these limitations by providing a means whereby gene products frompathogenic agents can be expressed in virtually unlimited quantities ina non-pathogenic form. Surface antigens from such viruses as influenza(1), foot and mouth disease (2), hepatitis (3), vesicular stomatitisvirus (4), rabies (5), and herpes simplex viruses (6) have now beenexpressed in E. coli and S. cerevisiae, and, in the future, promise toprovide improved subunit vaccines. It is clear, however, that theexpression of surface antigens in lower organisms is not entirelysatisfactory in that potentially significant antigenic determinants maybe lost by virtue of incomplete processing (e.g., proteolysis,glycosylation) or by denaturation during the purification of the clonedgene product.

This is particularly true in the case of membrane proteins, which,because of hydrophobic transmembrane domains, tend to aggregate andbecome insoluble when expressed in E. coli. Cloned genes coding formembrane proteins can be expressed in mammalian cells where the hostcell provides the factors necessary for proper processing, polypeptidefolding, and incorporation into the cell membrane (7,8). While thesestudies show that membrane proteins can be expressed on the surface of arecombinant host cell, and, for example (8), that a truncated membraneprotein lacking the hydrophobic carboxy-terminal domain can be slowlysecreted from the host cell rather than be bound to it, it is not clearthat either the membrane-bound protein thus expressed or the truncatedprotein thus secreted will be able to act, in fact, to raise antibodieseffective against the pathogen from which the protein is derived.

Herpes Simplex Virus (HSV) is a large DNA virus which occurs in tworelated, but distinguishable, forms in human infections. At least fourof the large number of virus-encoded proteins have been found to beglycosylated and present on the surface of both the virion and theinfected cells (9). These glycoproteins, termed gA/B, gC, gD, and gE,are found in both HSV type 1 (HSV1) and HSV type 2 (HSV2), while in thecase of HSV 2, an additional glycoprotein (gF) has been reported to befound (10). Although their functions remain somewhat of a mystery, theseglycoproteins are undoubtedly involved in virus attachment to cells,cell fusion, and a variety of host immunological responses to virusinfection (11). Although HSV 1 and HSV 2 show only ˜50 percent DNAsequence homology (12), the glycoproteins appear to be, for the mostpart, type-common. Thus, gA/B, gD, and gE show a large number oftype-common antigenic determinants (13-16), while gC, which waspreviously thought to be completely type-specific (17,18), has also beenfound to possess some type-common determinants. Type specific antigenicdeterminants can, however, be demonstrated using monoclonal antibodiesfor some of the glycoproteins (10,19), showing that some amino acidchanges have occurred since HSV1 and HSV2 diverged.

One of the most important glycoproteins with respect to virusneutralization is gD (11). Considerable evidence has been adducedstrongly suggesting that the respective gD proteins of HSV-1 and HSV-2are related. For example, recombination mapping has localized therespective genes to colinear regions in both virus genomes. Amino acidanalysis showed gross homology between the two proteins. The gD proteinsinduce neutralizing antibodies to both type 1 and type 2 viruses in atype-common manner (19-21). In addition, most monoclonal antibodiesgenerated to these glycoproteins are type-common, also suggesting a highdegree of structural relatedness between the two types of glycoproteins(20). Some monoclonal antibodies, however, were found to reacttype-specifically, suggesting significant differences between theproteins (19). Peptide maps of the proteins also unambiguously revealedsuch differences (22a). These results although suggesting-that thesepolypeptides are related, are insufficient to indicate exactly how closethe relationship is.

In order to examine the nature of the type-commonality of HSV-1 andHSV-2 gD proteins, the DNA sequences of the gD genes from HSV1 and HSV2were determined. The derived amino acid sequences showed similarity. Theresultant derived protein sequences were also analyzed for structuraldifferences by using a program designed to determine hydrophobic andhydrophilic regions of the protein. This analysis demonstrated a highdegree of conservation on a gross structural level. Although severalamino substitutions were found between the two glycoproteins, the vastmajority of these substitutions were conservative, suggesting animportant structural requirement of this glycoprotein to the virus.

In contrast to HSV-1, HSV-2 appears to encode yet another glycoprotein,termed gF (22b,10,22c,22d). Although the HSV-2 gF had an electrophoreticmobility which was much faster than HSV-1 gC, mapping studies withrecombinant viruses revealed that this protein was encoded by a regionof the HSV-2 genome which was approximately colinear with the gene forHSV-1 gC (22c,22d). In addition, it has been recently demonstrated thata monoclonal antibody against HSV-2 gF will cross-react weakly withHSV-1 gC (22f) and that a polyclonal antiserum made against HSV-1 virionenvelope proteins precipitated gF (22d), suggesting a possiblestructural homology between the two glycoproteins. Thus, it appearedthat a possible homologue to HSV-1 gC was the HSV-2 gF protein. Thisrelationship was investigated in accordance with the present invention.

To examine the relatedness between HSV-1 and HSV-2, it has beendetermined herein that a DNA sequence of a 2.29 kb region of the HSV-2genome is colinear with the HSV-1 gC gene. Translation of a large openreading frame in this region demonstrates that a protein which hassignificant homology to HSV-1 gC is encoded in this region. It issuggested that this region encodes the HSV-2 gF gene and that the gFprotein is the HSV-2 homologue of HSV-1 glycoprotein C.

SUMMARY OF THE INVENTION

One specific embodiment of the invention relates to a vaccine based onthe gD protein. In the light of this information about the structure ofthe gD protein, as described more fully herein, it was decided toexpress the gD protein DNA in mammalian cells to see whether such waspossible, and if possible, whether the expressed protein would bind tothe host cell membrane, and whether a truncated form of protein lackingthe membrane-binding domain would be secreted from the host cell, and ineither of the latter cases whether the expression product proteins couldraise antibodies effective against HSV-1 and/or HSV-2. As the resultsherein demonstrate, these objects have been achieved. In particular, theinvention provides using these proteins obtained by recombinant DNAprocesses as components in a vaccine effective against HSV-1 and HSV-2viruses. Thus provided are protective vaccines against occurrence ofherpes infection and of reduction in frequency and severity of herpesinfection recurrence in individuals already infected.

Another specific embodiment relates to another class of glycoproteinsobtained by recombinant DNA processes useful as components in a vaccineagainst HSV-1 and/or HSV-2 viruses. Specifically, such glycoproteinclass includes HSV-1 gC (effective against HSV-1), HSV-2 gF (moreproperly referred to as an HSV-2 gC), effective against HSV-2, orcombinations of the two proteins, effective against both viruses. Othersuch glycoproteins include gA, gB, and gE. It is believed that a vaccinebased upon the combined gC and gD glycoproteins would be significantlymore effective as a vaccine than either glycoprotein alone.

To further summarize, the present invention involves a vaccinecomprising a polypeptide with antigenic determinants capable ofspecifically raising complementary antibody against HSV-1 and HSV-2viruses. In one embodiment, the polypeptide is functionally associatedwith the surface membrane of a recombinant host cell capable of itsproduction. In a typical instance, such functional association comprisesa binding of the polypeptide with the surface membrane so that thepolypeptide projects through the membrane. The recombinant cell line isderived from a stable, continuous line.

In another embodiment, the vaccine comprises a polypeptide with the sameantigenic determinants, but which is not functionally associated withthe surface membrane. As set out in more detail below, one suchpolypeptide is a truncated, membrane-free derivative of a membrane-boundpolypeptide. The derivative is formed by omission of a membrane-bindingdomain from the polypeptide, allowing it to be secreted from therecombinant host cell system in which it has been produced.

In another embodiment, the polypeptide is formed first in functionalassociation with a surface membrane and thereafter the polypeptide isdissolved, preferably in a non-ionic surfactant, to free the polypeptideof the membrane.

As used herein, the term "recombinant" refers to cells which have beentransfected with vectors constructed using recombinant DNA technologyand thus transformed with the capability of producing the polypeptidehereof. "Functional association" is meant being bound to the membrane,typically by projecting to both sides of the membrane, in such manner asto expose antigenic determinants folded in a native conformationrecognizable by antibody elicited against the native pathogen."Membrane-bound" in reference to polypeptides hereof refers to a classof polypeptides ordinarily produced in eukaryotic cells andcharacterized by having a signal sequence which is believed to assistits secretion through various cell membranes as well as amembrane-binding domain (usually hydrophobic in nature and occurring atthe C-terminal end) which is thought to preclude its complete secretionthrough the cell membrane. As such, it remains functionally associatedor bound to the membrane. This invention is particularly directed to theexploitation of those membrane-bound polypeptides associated withpathogenic organisms, e.g., herpes virus.

As used herein, the terms "HSV-2 gF", "HSV-2 gC" and "gC-2" are usedinterchangeably to refer to a glycoprotein portion of HSV-2 which ishighly homologous with HSV-1 gC and which is capable of raisingsufficient antibodies to be useful as a vaccine.

Once the antigenic determinants of the polypeptides of the presentinvention are provided by functional association with the surfacemembrane, thereafter, the membrane may be removed from the polypeptideswithout destroying the antigenic characteristics. Thus, for example, themembrane-bound polypeptide may be removed from the membrane bysolubilization with a suitable solution, preferably one containing anon-ionic surfactant, to remove the polypeptide from the membrane. Anadvantage of doing this is to isolate the polypeptide from extraneouscellular material, raising potential potency in its use in a vaccine. Atechnique for removing the membrane from the polypeptide is describedbelow.

In another embodiment, membrane-free preparations may be obtained bycreation of a secretion system. As described in more detail below, suchsecreted polypeptide possess at least some of the antigenic sitesnecessary for antibody stimulation.

In the accompanying drawings:

FIGS. 1A and 1B show the DNA and deduced amino acid sequences of theHSV-1 and HSV-2 gD genes and surrounding flanking regions;

FIG. 2 shows a hydropathy analysis of the gD proteins from HSV-1 andHSV-2 proteins;

FIG. 3 is a diagram of the plasmid pgD-dhfr, constructed for theexpression of a membrane-bound form of HSV-1 glycoprotein D;

FIG. 4 shows the result of labelling of gD12 cells with human antibodiesagainst HSV, (A) being a visualization with phase contrast optics, (B) afluorescence image of the same cells;

FIG. 5 shows radioimmunoprecipitations of cloned gD from the gD12 cellline hereof and native gD from HSV-1 infected human cells;

FIG. 6 shows the binding of human anti-HSV antibodies to gD12 cells andthe parental CHO cell line.

FIG. 7 is a schematic representation of HSV-1 gD protein and illustratesthe locations of signal sequence and membrane-binding domain.

FIG. 8 is a diagram of the expression plasmid pgDtrunc-dhfr for asecreted form of HSV-1 gD protein.

FIG. 9 shows radioimmunoprecipitations from the gD10.2 cell line hereof.

FIG. 10 shows radioimmunoprecipitations from preamplified and amplifiedgD10.2 cell lines.

FIG. 11 demonstrates the degree of amplification achieved with the Mtxamplified gD10.2 cell line.

FIG. 12 shows the fragments of pgC₂ Sal2.9 which were subjected to DNAsequence analysis.

FIG. 13 shows the DNA sequence derived from pgC₂ SA12.9 compared withthe DNA sequence of the HSV-1 gC region.

FIG. 14 illustrates southern blot analysis of HSV-2 genomic DNA and pgC₂Sal12.9 DNA.

FIG. 15 illustrates translation of the HSV-2 large open reading frameand comparison with the HSV-1 gC amino acid sequence.

FIG. 16 illustrates hydropathy analysis of the HSV-1 gC protein and theHSV-2 major open reading frame protein.

DETAILED DESCRIPTION (EXAMPLES) Example 1

Example 1 relates to gD protein.

Virus Growth and Viral DNA Isolation

HSV1 (strain Hzt) and HSV2 (strain G) were grown on Hep 2 cells at 37°C. and at 33° C., respectively. The viral DNA was isolated from infectedcell cultures by proteinase K digestion and CsCl banding (23).

Cloning of the gD Genes of HSV1 and HSV2

Previous mapping and cloning studies had localized the HSV1 gD gene to a˜6.6 kb Bam HI fragment (6,24). HSV1 DNA was cleaved with Bam HI, andthe 6-7 kb region was isolated by agarose gel electrophoresis. Thisfragment was ligated into Bam HI-digested pBR322, and the resultantmixture was used to transform E. coli strain 294 (ATCC No. 31446). Theampicillin resistant, tetracycline sensitive plasmids were screened forthe proper HSV1 fragment by restriction enzyme digestion. The correct gDcontaining Sst 1 fragment was subcloned into Sst1-digested plasmid pFM3(European Patent Application Publication No. 0068693; 5 Jan. 1983).

Although the gD gene from HSV2 was previously mapped by recombinationwith HSV1, the exact location of this gene was unknown. Therefore, an˜10 kb Hind III fragment from the small unique region of the HSV2 genome(4) was ligated into the Hind III site of the bacteriophage lambdacloning vector 590 (25). In vitro packaged phage were plated at lowdensity and screened by the Benton-Davis procedure with a ³² P-labeledsubclone of the gD gene from HSV1 (26). Positively hybridizing plaqueswere grown, the DNA isolated, and the gD gene localized by Southernblotting and hybridizaton with the ³² P-labeled HSV1 gD gene (27). Thepositively hybridizing, HSV2 gD containing fragments were subcloned intothe plasmid pUC9 (28).

DNA Sequence Determination and Computer Analysis

Various fragments from the HSV1 and HSV2 gD genes were subcloned intothe ml3 phage vector mp9 (29), and were sequenced by thedideoxynucleotide method of Sanger (30).

The nucleotide sequences were analyzed using the HOM program (31). Thehydropathy of the deduced protein sequence was analyzed using a width of12 and a jump of 1 (31a). Cloning of the gD Regions from HSV1 and HSV2

Other studies had localized the HSV1 gD gene to the 6.6 kb Bam HI Jfragment according to the nomenclature of Roizman (6,12,24). Isolationand sequencing of part of this fragment showed that this fragmentcontained the HSV1 gD gene. Since one might expect that the DNAsequences of the HSV1 gD gene would be relatively homologous to the HSV2gD gene, this fragment was used as a probe for the isolation of the gDgene from the HSV2 genome.

Since most of the genes from the HSV1 and HSV2 genomes appear to mapcolinearly (35), the region from the small unique region of the HSV2genome which corresponded to the HSV1 gD region (the Hind III L fragment(12)), was cloned into a lambda phage vector. Screening of the resultantplaques with a ³² P-labeled HSV1 gD gene subclone revealed positivelyhybridizing plaques, suggesting that there was indeed nucleic acidsequence homology between the two virus genomes in this region.Isolation of the phage DNA and subsequent Southern blot analysisrevealed the region of this fragment which corresponded to the gD gene.This region was subcloned for DNA sequence analysis.

The Coding Regions

FIG. 1 illustrates the two gD DNA sequences compared with the HOMprogram (31). Nucleotide number 1 is chosen as the A of the ATGinitiator methionine. Gaps have been introduced by the HOM computerprogram to maximize the sequence homologies (31). Nucleotide differencesare shown by the symbol (*), while amino acid differences are shownboxed. Amino acid differences between the HSV1 gD sequence reportedhere, determined for the Hzt strain of HSV1, and that reported by Watsonet al. (6) for the Patton strain, are depicted by the symbol (+). Thestart of HSV1 gD gene transcription, shown by an arrow, is from Watsonet al. (32). Possible N-linked glycosylation sites are shown shaded. Twopossible "TATA" sequences are shown 5' to the start of gD transcription,while a third possible "TATA" sequence is shown 5' to a second openreading frame at the 3' end of the HSV2 sequence. Two regions ofnon-coding sequence homology should be noted 5' to the gD genes and 5'to the second open reading frame from the HSV2 sequence.

The Hydropathy of gD Proteins

The hydropathy of each glycoprotein was analyzed using the programdeveloped by Hopp et al. (31a). As shown in FIG. 2, a hydrophobictransmembrane domain exists at the 3'-end of the gene. Twelve amino acidlong stretches were analyzed, and the average hydropathy was calculated.Residue differences between the two glycoproteins are shown, withconservative changes marked (*) and non-conservative changes marked (+).A) HSV1 gD protein hydropathy, B) HSV2 gD protein hydropathy.

The DNA sequence analysis demonstrates that the HSV1 and HSV2 gDproteins are 80 percent homologous. The majority of the differencesfound between these two proteins were in the amino and carboxy terminalregions. The amino-terminal region of these proteins contains a highlyhydrophobic region which contains an arginine residue near theamino-terminal methionine. This hydrophobic domain is the signalsequence which is characteristic of secreted and membrane-bound proteinsand which presumably functions to direct at least a portion of theprotein into the lumen of the endoplasmic reticulum (33). A comparisonof the first twenty amino-terminal amino acids showed that there were atotal of 12 differences between the type 1 and type 2 genes. Virtuallyall of the differences, however, are conservative since they encodeother hydrophobic amino acids. The exceptions are the gly-argreplacement at residue 3 and the arg-gly replacement at residue 7.Although these replacements are not conservative, they do not change thenet structure of the signal domain. Both genes maintain a positivelycharged residue within the first 10 amino acids.

The hydropathy plot in FIG. 2 revealed a hydrophilic carboxy-terminaldomain preceded by a hydrophobic region. This structure ischaracteristic of membrane-bound glycoproteins and has been previouslyfound in other viral surface antigens (5,34). Its function is to anchorthe protein in the cellular and viral membranes and, as such, performsan important role for virus infection. Twelve amino acid changes in thisregion of the gD proteins from residues 333 to 362 were found, most ofwhich are conservative. This suggests that the only criterion for theamino acids in this region is that they be predominantly apolar in orderto span the lipid bilayer. In addition, the region after the membranedomain (residues 363-375), which probably serves to anchor the proteinin the membrane (33), shows 5 changes in its first 13 residues followedby a long homologous stretch. This result suggests that the initial10-15 residues in the carboxy-terminal hydrophilic domain may only servean anchoring function and therefore only need to be charged, while thesubsequent 23 residues may serve some other function important to the gDprotein specifically.

Although many other amino acid changes are found throughout these twoproteins, the vast majority of the changes are conservative. This factis underlined by the structure revealed by the hydropathy program shownin FIG. 2. As can be seen in this comparison, the two glycoproteins showvery similar plots. The amino acid changes which are not conservative donot appear to change the hydropathy of the protein.

Expression of the HSV-1 gD

In order to establish a permanent membrane-bound gD producing cell line,the gD containing fragment was ligated (FIG. 3) into a mammalianexpression vector (36) containing the selectable marker, dihydrofolatereductase (dhfr). FIG. 3 shows a diagram of the plasmid, pgD-dhfr,constructed for the expression of HSV-1 glycoprotein D. The expressionplasmid consisted of the origin of replication and the β-lactamase gene(amp^(r)) derived from the E. coli plasmid pBR322 (37), a cDNA insertencoding mouse dhfr (36,38) under control of the SV-40 early promoterand a 4.6 kb HindIII to Bam HI fragment containing the gD gene alsounder control of the SV-40 early promoter. The HindIII end of thisfragment lies 74 bp to the 5' side of the initiator methionine codon andincludes the mRNA cap site. The HindIII site lies 250 bp to the 3' sideof the Goldberg-Hogness box of the SV-40 promoter. The coding region ofthe gD-containing fragment is 1179 bp long and adjoins a large (1.9 kb)3' region which contains at least part of the glycoprotein E gene (24,32), a translational stop codon, and a polyadenylation site.

The plasmid pgD.dhfr was constructed as follows: The 4.6 kilobase HindIII-Bam HI fragment containing the entire gD coding sequence wasisolated from the Bam HI fragment cloned from the HSV 1 genome (seeabove). The 2.8 kilobase Hind III-Sal I fragment containing an SV40origin-early promoter and the pBR322 ampicillin resistance gene andorigin of DNA replication were isolated from the plasmid pEHBal 14. The2.1 kilobase Sal I-Bam HI fragment containing a murine dihydrofolatereductase cDNA clone-under the control of a second SV40 origin-earlypromoter was isolated from the plasmid pE348HBV E400D22 (36). Thesethree fragments were ligated together in a triple ligation using T4 DNAligase, and the resultant mixture was used to transform E. coli strain294. The resultant colonies were grown and the plasmid DNA screened bydigestion with Sac III. The correct DNA construction pgd.dhfr (FIG. 3)was used for further transfection studies.

The plasmid was introduced into Chinese Hamster Ovary cells (CHO)deficient in the production of dhfr (39) using a calcium phosphateprecipitation method (40). Colonies capable of growth in media lackinghypoxanthine, glycine, and thymidine were obtained and nine dhfr⁺ cloneswere analyzed. Of these, gD could be detected in five colonies usinganti-HSV-1 antibodies in radioimmunoprecipitation and indirectimmunofluorescence assays. One of the five lines (gD12) was designatedfor further study. In order to characterize the cloned gD gene product,gD12 cells were metabolically labeled with ³⁵ S-methionine or ³H-glucosamine and analyzed by radioimmunoprecipitation. The procedureused was as follows: Cells were grown in Ham's F12 medium (GIBCO)supplemented with 7 percent commercially dialyzed fetal bovine serum(GIBCO), penicillin (100 u/ml), and streptomycin (100 u/mil). When thecultures were approximately 80 percent confluent, the medium wasremoved, the cells were washed twice with phosphate buffered saline(PBS), and labeling medium (Dulbecco's modified Eagle's mediumcontaining either one-tenth the normal concentration of methionine orglucose) was added to a final concentration of 0.064 ml/cm². Either ³⁵S-methionine (SJ.204, Amersham Int.) (50-75 μCi/ml) or 3H-glucosamine(100 μCi/ml) was added and the cells were grown for an additional 18-20hr. After labeling, the medium was harvested and the cells were washedtwice in PBS, and removed from the culture dish by treatment with PBScontaining 0.02 percent EDTA. The cells were then solubilized in lysisbuffer consisting of: PBS, 3 percent NP-40, 0.1 percent bovine serumalbumin, 5×10⁻⁵ M phenylmethylsulfonyl fluoride, and 0.017 TIU/ml ofapoprotinin and the resultant lysate was clarified by centrifugation at12,000× g. For immunoprecipitation reactions cell lysates were diluted3-fold with PBS and aliqouts (typically 180 μl) were mixed with 2-5 μlof antisera and incubated at 4° C. for 30 min. Immune complexes werethen adsorbed to fixed S. aureus cells by the method of Kessler (40a)and were precipitated by centrifugation at 12,000× g for 30 s. The S.aureus cells were then washed 3 times with wash buffer (PBS, 1 percentNP-40, 0.3 percent sodium dodecyl sulfate), and the immune complexeswere eluted with 20 μl of polyacrylamide gel sample buffer (62.5 mMTris-HCl buffer, pH 6.8 containing 10 percent glycerol, 5 percent2-mercaptoethanol, 0.01 percent bromophenol blue) at 90° C. for 3 min.After centrifugation for 30 s the supernatants were applied to 10percent polyacrylamide slab gels according to the method of Laemmli(45).

FIG. 4A compares autoradiographs obtained with the gD12 cell line andHSV-1 infected cells: control immunoprecipitation from the gD12 celllysate with normal rabbit serum (lane 1); immunoprecipitation of nativegD grown in HEL cells (lane 2) and A549 cells (lane 3) with themonoclonal anti-gD antibody, 55-S (41); immunoprecipitation of cloned gDfrom the gD12 cell lysate with polyclonal rabbit antibodies (Dako Corp.)to HSV-1 (lane 4), and the monoclonal antibody, 55-S (lane 5);immunoprecipitation of cloned gD from the gD12 cells metabolicallylabeled with ³ H-glucosamine with polyclonal rabbit anti-HSV-1antibodies (lane 6).

It is seen (lanes 4 and 5) that a diffuse band of 59-60 kd wasspecifically precipitated from the gD12 cell line using either rabbitanti-HSV-1 antibodies or the monoclonal anti-gD antibody, 55-S, specificfor the HSV-1 protein (41). This molecular weight agrees well with thatreported for gD isolated from HSV-1 infected KB cells (42). It is seenthat the same monoclonal antibody precipitated proteins of similar butdifferent molecular weights from HSV-1 infected human cell lines. Themajor product precipitated from the A549 human lung carcinoma cell line(lane 2) was 53 kd and that precipitated from the human embryonic lungcell line (HEL) was 56 kd (lane 3). Previous studies (43) have shownthat the molecular weight of HSV glycoproteins varies depending on thehost cell and that these differences are due to differences inglycosylation. To determine whether the gD protein produced in CHO cellswas, in fact, glycosylated, the cells were metabolically labeled with ³H-glucosamine. Because bands of identical molecular weights (lanes 5 and6) were precipitated after metabolic labeling with ³⁵ S-methionine or3H-glucosamine, we concluded that the gD protein produced in CHO cellsis glycosylated.

The human cell lines A549 (ATCC CCL 185) and HEL 299 (ATCC CCL 137) weregrown to confluence in 3.5 cm tissue culture dishes and infected withHSV-1 at multiplicity of 10 pfu per cell. Virus infected cells werelabeled by a method similar to that described by Cohen et al. (44). 4 hrafter infection the medium was removed and the cells were washed oncewith fresh medium (Dulbecco's modified Eagle's medium) and once withphosphate-buffered saline (PBS). Fresh medium containing one-tenth thenormal concentration of methionine was then added to the cells alongwith ³⁵ S-methionine Amersham, International) to a final concentrationof 75 μCi per ml of medium. The cells were grown an additional 20 hr andthen harvested by treatment of washed cells with PBS containing EDTA(0.02 percent). Viral proteins were solubilized in lysis bufferconsisting of PBS, 3 percent NP-40, 1 percent bovine serum albumin,5×10-5M phenylmethylsulfonyl fluoride, and 0.017 TIU/ml of apoprotinin.The resultant lysate was clarified by centrifugation at 12,000× g in amicrocentrifuge. For immunoprecipitation reactions the cell or viruslysates were diluted 3-fold with phosphate buffered saline, mixed with2-5 μl of the appropriate antiserum and incubated for 30 min at 4° C.Antibody-antigen complexes were removed from the reaction medium by theaddition of 25 μl of a 10 percent solution fixed S. aureus (Kessler(40a)) and were precipitated by centrifugation at 12,000× g for 30 s.The S. aureus cells were then washed 3 times with wash buffer (PBS, 1percent NP-40, 0.3 percent sodium dodecyl sulfate), and the cellssuspended in 20 μl of polyacrylamide gel sample buffer (10 percentglycerol, 5 percent 2-mercaptoethanol, 0.0625M in pH 6.8 Tris buffer,0.01 percent bromophenol blue) and incubated at 90° C. for 3 min. Aftercentrifugation (12,000×g) for 30 s the supernatants were applied to 10percent polyacrylamide slab gels (45).

To further explore the post-translational processing of cloned gD,pulse-chase studies were conducted. FIG. 4B shows immunoprecipitation ofcloned gD from gD-12 cells with rabbit anti-HSV-1 antibodies (Dako,Corp.) at various times after pulse labeling with ³⁵ S-methionine. FIG.4B shows a pulse labelling of the gD12 cells. In these studies, cellswere grown to confluence in 10 cm tissue culture dishes and labeled with³⁵ S-methionine as described above with the exception that the labelingreaction was carried out for 15 min. on ice, the cells washed 3 timeswith fresh medium, and then returned to the incubator and incubated at37° C. for various times. The reactions were terminated by washing thecells in cold phosphate-buffered saline and solubilizing the cells asdescribed above. Proteins were immunoprecipitated at the following timesafter pulse labeling: lane 1, 5 min; lane 2, 15 min; lane 3, 30 min;lane 4, 60 min; lane 5, 120 min. The precursor form of gD with amolecular weight of 51 kd was specifically precipitated from the gD12cell line 5 min after a pulse with ³⁵ S-methionine, and this precursorchased into the higher molecular weight form (59 kd) after approximately60 min. From these studies we estimate the half-time for thispost-translational event to be approximately 45 min. Theprecursor-product relationship between the 51 kd band and 59 kd bandclosely resembles that reported for virus produced gD (14,42,46,47) andthe kinetics of this process are similar to those described by Cohen etal. (42). In virus infected cells the difference in molecular weightsbetween the precursor and the product has been attributed to bothN-linked and O-linked oligosaccharides (48).

To determine whether gD was exported to the cell surface, indirectimmunofluorescence studies were conducted. In these studies rabbit,mouse, and human anti-HSV antibodies were reacted with unfixed cellsunder conditions which do not permeablize the cell membrane (49). gD12cells and the parental CHO cells (1:1 ratio) were plated onto glasscoverslips (2.2×2.2 cm) and grown until the cells were approximately 60percent confluent. Human serum known to contain antibodies to HSV-1 (50)was diluted forty-fold with phosphate buffered saline (PBS) and 100 μlwas pipetted onto washed cells and was incubated for 30 min. at roomtemperature in a humidified chamber. The cells were immersed 3 times inPBS to wash away unbound antibody and then were incubated with 100 μl of20-fold diluted tetramethylrhodamine isothiocyanate-labeled goatanti-human IgG antibodies (Cappel Laboratories) for an additional 30min. The unbound labeled antibody was washed away with PBS and the cellswere dehydrated in ice cold 50 percent ethanol and 100 percent ethanoland rehydrated with glycerol on a microscope slide (49). The cells werethen viewed under phase-contrast and fluorescence optics in fluorescencemicroscope (Zeiss). FIG. 5 shows: A, gD12 and CHO cells viewedvisualized with phase contrast optics; B, fluorescence image of the samecells as in A. Comparison of the phase-contrast images with thefluorescence images (FIG. 5) showed that the gD12 cells were heavilylabeled, while the parental CHO cells bound little or no labeledantibody. In control experiments with normal mouse sera, normal rabbitsera, or human sera known to be negative for HSV antibodies, no specificlabeling of the cells could be detected. These studies suggested thatthe gD was exported to the cell surface. Experiments with CHO and gD12cells fixed prior to labeling with agents known to permeablize the cellmembrane (methanol or acetone) gave a different labeling pattern. Inthese studies we observed heavy perinuclear labeling of the gD12 cellswith anti-HSV-1 antibodies, and no specific labeling of the CHO cells.

In order to determine whether gD12 cells expressed antigenicdeterminants relevant to human HSV-1 and HSV-2 infections, the bindingof antibodies from individuals known to possess anti-HSV-1 or anti-HSV-2antibodies (50) was examined. Radioimmunoprecipitation of lysates frommetabolically labeled gD12 cells gave results comparable to thoseobtained with rodent anti-HSV sera (FIG. 4). Similarly, human anti-HSV-1sera gave specific labeling of gD12 cells in an indirectimmunofluorescence assay (FIG. 5) and did not label the parental CHOcell line. Taken together, the results obtained with various rodentanti-HSV-1 and HSV-2 antisera, monoclonal anti-gD antibodies and humananti-HSV antisera provide evidence that gD expressed on the surface ofgD12 cells possesses a number of antigenic determinants in common withthe native virus and that the structure of these determinants is notdependent on interactions with other HSV-1 proteins. The fact that oneof the monoclonal antibodies tested (1-S) is known to neutralize HSV-1in vitro (41) and in vivo (51) demonstrates that the gD produced in CHOcells possesses at least one of the neutralizing antigenic determinantsin common with the native virus.

In order to have a quantitative measure of the binding of anti-HSVantibodies to gD12 cells, an enzyme-linked immunosorbtion assay (ELISA)was developed (52). In these studies gD12 cells and CHO cells wereplated and chemically fixed into alternate wells of 96 well microtitertissue culture plates. Various antisera known to possess antibodies toHSV were then serially diluted and allowed to react with the fixedcells. At the end of the assay, the absorbance in each well was measuredand normal binding curves were constructed. The specific binding ofantibodies to the gD12 cells was determined by subtracting the valuesobtained with the parental CHO cells from those obtained from the gD12cells. Specific binding by high titer sera could be detected atdilutions of 1:10,000.

We compared serum titers determined using the gD12 cell ELISA assay withanti-HSV-1 and anti-HSV-2 titers determined by conventional methods.Human sera previously titered (50) against HSV by conventional assays,i.e., inhibition of hemagglutination (IHA) or complement fixation (CF),was serially diluted into wells of microtiter plates containing eithergD12 cells or the parental CHO cell line and the binding of anti-gDantibodies was monitored in an ELISA assay. gD12 cells and the parentalCHO cells were seeded into alternate wells of 96 well microtiter tissueculture plates (Falcon Labware) and were grown to confluence in F12medium (GIBCO) containing 10 percent fetal bovine serum. The cells werewashed three times with phosphate-buffered saline (PBS) and then werechemically fixed with 0.0625 percent glutaraldehyde in PBS. The cellswere again washed three times with PBS and stored until needed at 4° inPBS containing 1 percent bovine serum albumin, 100 mM glycine 1 mM NaN₃.To measure anti-gD antibody titers, the cells were washed with PBS, andserially diluted antisera was allowed to react with the fixed cells (50μl final volume) for 1 hr at room temperature. Unbound antibody waswashed away and the cells were incubated with 50 μl of 1:2000 dilutedgoat anti-human IgG coupled to horseradish peroxidase (Tago, Inc.). Theenzyme-linked antibody was allowed to react for one hour at roomtemperature, and the cells were then washed three times with PBS. Afterincubation, the peroxidase substrate, o-phenylene diamine, was added(200 μl) and the reaction was allowed to proceed for 10 min. Thereaction was terminated by the addition of 2.5M H₂ SO₄ (50 μl) and theabsorbance of the reaction medium from each well was determined with anautomated plate-reading spectrophotometer (Titertek). In FIG. 6, theserum represented by the open and closed circles exhibited a HSV-1 CFtiter of 128 and HSV-1 and HSV-2 IHA titers of 4096. The serumrepresented by open and closed squares exhibited a HSV-1 CF titer of <8and HSV-1 and HSV-2 IHA titers of <8. A, closed circle and closed squareindicates binding to gD12 cells; open circle and open square indicatesbinding to CHO cells. B, closed circle and closed square represents thespecific binding to gD12 cells calculated by subtraction of the valuesin A. In FIG. 6 it can be seen that a serum with a high anti-HSV titerdetermined by conventional assays gave a high ELISA titer, while anotherserum with low anti-HSV titers gave no detectable binding in the gD12ELISA.

The studies described demonstrate that stable cell lines constitutivelyexpress on their surface a transfected gene product which binds withantibodies generated by herpes virus infection.

Immunization of mice with gD12 cells.

Twenty female BALBIc mice (5 weeks of age) were obtained from SimonsenLaboratories (Gilroy, Calif.). The mice were divided into two groups of10 mice each: an "experimental" group and a "control" group. Each mousein the experimental group was injected with gD12 cells known to expressHSV-1 glycoprotein D on their surface. Each mouse in the control groupwas injected with the parental Chinese hamster ovary cell line (CHOcells) from which the gD12 cell line was derived. For immunization ofmice both types of cells were grown to confluence in 15 cm tissueculture dishes. The CHO cells were grown in Hams F12 medium (GIBCO)supplemented with 7 percent commercially dialyzed fetal bovine serum(GIBCO), penicillin (100 U/ml), and streptomycin (100 U/ml). The gD12cells were grown in the same medium lacking glycine, hypoxanthine, andthymidine. To harvest the cells, each dish was washed twice with 15 mlof phosphate buffered saline (PBS) and then treated with 15 ml of PBScontaining 0.02 percent EDTA. After 15-20 min. the cells were thenremoved from the dish and pelleted by centrifugation for 5 min. at fullspeed in a clinical centrifuge (IEC model CL clinical centrifuge, rotormodel 221). The supernatant was discarded and the cells were resuspendedin PBS to a final concentration of 1 ml PBS per each 15 cm dish ofcells. Each mouse was then injected with 0.5 ml of cell suspension(˜5×10⁶ cells) distributed as follows: 0.25 ml injectedinterperitoneally, and 0.25 ml injected subcutaneously in the loose skinof the back of the neck. The mice were then boosted twice with freshcells (prepared as described above) on day 38 and day 55 after theinitial immunization. Mice were bled via the tail vein on day 68 toobtain sera for in vitro neutralization studies. Mice were challengedwith HSV-1 (Maclntyre strain) on day 70. The virus challenge entailed aninterperitoneal injection of 2×10⁷ pfu of virus into each mouse. Themice were scored daily for mortality and every other day for weightchange and the onset of paralysis. All of the mice in the control groupdied within 7 days of the virus challenge, while all of the experimentalmice were protected and showed no sign of infection. These studiesconclude that immunization with the gD12 cells protect from a lethalHSV-1 virus challenge.

A variety of transfection schemes are possible, of course, using avariety of selectable markers. For example, mouse L cells can beusefully transfected using a mutant dhfr gene as a selectable marker.The gD gene was transfected into such cells via a vector harboring sucha marker. In principle, the strategy which we have described could beapplied to any situation where the expression of a membrane protein isdesired.

Expression of a Truncated Form of the gD Gene

The foregoing description relates to the production of membrane-bound gDprotein. However, as discussed above in relation to FIG. 2, analysis ofthe amino acid sequences of the gD protein of HSV-1 and HSV-2 identifiedin each case a hydrophobic/hydrophilic carboxy-terminal membrane bindingdomain (FIG. 7).

A Schematic Diagram of the HSV 1 Glycoprotein D (gD)

Hydrophobic (shaded) and hydrophilic (marked+) regions of the proteinwere determined from the hydropathy analysis (31a) of the gD proteinsequence derived from the gene sequence. Only those regions thought tobe important for membrane localization and binding are shown. Thefunctional domains are: a) the signal sequence (33), b) the hydrophobictransmembrane domain, and c) the charged membrane anchor. The threeputative N-linked glycosylation sites are shown by the letter G. Theexpression plasmid consisted of the pBR322 bacterial origin ofreplication and ampicillin resistance gene, a cDNA insert encoding themurine dihydrofolate reductase gene under the transcriptional control ofthe SV40 early promoter (53) and a Hind III-Hinf I fragment whichencodes the first 300 amino acids of gD under the transcriptionalcontrol of a second Sv40 early promoter. The Hind III site of thisfragment lies 74 bp to the 5' side of the initiator methionine of the gDgene. The Hind III site of the SV-40 early region vector (36) lies 250bp to the 3' side of the Goldberg-Hogness box of the SV40 promoter. TheHinf I site (blunted with Klenow DNA polymerase and 4 deoxynucleotidetriphosphates) is ligated to the Hpa I site of the 3' nontranslatedregion of the hepatitis B virus surface antigen gene (36). This methodis also useful for preparing a truncated HSV-2 gene. The resultantsequence creates a stop codon (TAA) immediately after amino acid 300 ofthe gD gene. The transcription termination and polyadenylation sites forthe truncated gD gene transcript are encoded by the 3' untranslatedregion of the hepatitis B surface antigen gene (36).

The plasmid pgDtrunc.dhfr was constructed as follows: The 2.9 kilobasegD-containing Sac 1 fragment was isolated from the Bam HI fragmentcloned from the HSV 1 genome (see above) in the plasmid pFM3 (see above)cut with Sac I. A 1.6 kilobase Hind III-Bst NI fragment containing theentire gD gene was subcloned into Hind III-Bst NI digested pFM42 (EPOApplication No. 68693). This plasmid was then cut with Hinf I, bluntedwith Klenow DNA polymerase and four deoxynucleotide triphosphates, andthen subsequently cut with Hind III. The 960 base pair Hind III-bluntHinf I fragment containing the truncated gD gene was isolated andligated to Hind III-Hpa I digested pEHBal14. The resultant construction(pgDCos-trunc) contained the truncated gD gene with the hepatitis Bsurface antigen gene at its 3 prime end. A 2.3 kilobase Hind III-Bam HIfragment containing the truncated gD gene was isolated frompgDCos-trunc. The 2.8 kilobase fragment containing the SV 40origin-early promoter and the pBR322 ampicillin resistance gene andbacterial origin of replication were isolated from the plasmid pEHBal14. The 2.1 kilobase fragment containing the murine dihydrofolatereductase cDNA clone under the transcriptional control of a second SV 40early promoter was isolated from the plasmid pE348HBVE400D22 (36). Thesethree fragments were ligated together with T4 DNA ligase, and theresultant mixture was used to transform E. coli strain 294. Plasmid DNAfrom the resultant colonies was screened with Sac III, and the correctconstruction pgDtrunc.dhfr (FIG. 8) was used for further transfectionstudies.

Plasmid pEHBal 14 was constructed by cleaving pE342ΔR1 (describedbelow), an SV40-hepatitis chimera, with Xba I, which cleaves once in thecoding region of the HBV surface antigen, and sequentially removingsequences surrounding this Xba I site by using nuclease Bal31. Theplasmid was ligated in the presence of the synthetic oligonucleotide5'-AGCTGAATTC, which joins the HBV DNA with a Hind III restriction site.

Resulting plasmids were screened for an Eco RI-Hind III fragment of ˜150b.p. pEHBal 14 was sequenced, which verified that a Hind III site hadbeen placed at a point just upstream of where the HBsAg initiation codonis normally found. This construction thus places a unique Hind III sitesuitable for cloning at a position where a highly expressed protein(HBsAg) initiates translation. Any putative signals necessary for highexpression of a protein should be present on this 5' leader sequence.

Plasmid pE342 which expresses HBV surface antigen (also referred to aspHBs348-E) has been described by Levinson et al, EPO Publication No.0073656, Mar. 9, 1983, which is incorporated herein by reference.(Briefly, the origin of the Simian virus SV40 was isolated by digestingSV40 DNA with Hind III, and converting the Hind III ends to EcoRI endsby the addition of a converter (AGCTGAATTC)). This DNA was cut with PvuII, and RI linkers added. Following digestion with Eco RI, the 348base-pair fragment spanning the origin was isolated by polyacrylamidegel electrophoresis and electroelution, and cloned in pBR322. Expressionplasmid pHBs348-E was constructed by cloning the 1986 base-pair fragmentresulting from EcoRI and BglII digestion of HBV (Animal Virus Genetics,(Ch. 5) Acad. Press, N.Y. (1980)) (which spans the gene encoding HBsAg)into the plasmid pML (Lusky et al., Nature, 293: 79 (1981)) at the EcoRI and Bam HI sites. (pML is a derivative of pBR322 which has a deletioneliminating sequences which are inhibitory to plasmid replication inmonkey cells). The resulting plasmid (pRI-Bgl) was then linearized withEco RI, and the 348 base-pair fragment representing the SV40 originregion was introduced into the Eco RI site of pRI-Bgl. The originfragment can insert in either orientation. Since this fragment encodesboth the early and late SV40 promoters in addition to the origin ofreplication, HBV genes could be expressed under the control of eitherpromoter depending on this orientation (pHBS348-E representing HBsexpressed under control of the early promoter). pE342 is modified bypartially digesting with Eco RI, filling in the cleaved site usingKlenow DNA ploymerase I, and ligating the plasmid back together, thusremoving the Eco RI site preceding the SV40 origin in pE342. Theresulting plasmid is designated pE342ΔR1.

The resultant sequence creates a stop codon (TAA) immediately afteramino acid 300 of the gD gene. The transcription termination andpolyadenylation sites for the truncated gD gene transcript are encodedby the 3' untranslated region of the hepatitis B surface antigen gene(36).

The resulting vector was transfected (40) into a dhfr⁻ CHO cell line(39), and a suitable clone gD10.2 selected which produced the truncatedgD protein and secreted it into the surrounding medium. The protein wasextracted from the medium and the cells were tested for immunogenicactivity. FIG. 9 shows the results of immunoprecipitations of intra- andextra-cellular ³⁵ S-methionine-labelled extracts.

Radioimmunoprecipitation of cell associated- and secreted-forms of gD.Cells were grown in Ham's F12 medium (GIBCO) supplemented with 7 percentcommercially dialyzed fetal bovine serum (Gibco), penicillin (100 u/ml),and streptomycin (100 u/ml). When the cultures were approximately 80percent confluent, the medium was removed, the cells were washed twicewith phosphate buffered saline (PBS), and labeling medium (Dulbecco'smodified Eagle's medium containing one-tenth the normal concentration ofmethionine) was added to a final concentration of 0.05 ml/cm2. ³⁵S-methionine (SJ.204, Amersham Int.) was added to a final concentrationof 50-75 μCi/ml and the cells were grown for an additional 18-20 hr.After labeling, the medium was harvested and the cells were washed twicein PBS, and removed from the culture dish by treatment with PBScontaining 0.02 percent EDTA. The cells were then solubilized in lysisbuffer consisting of: PBS, 3 percent NP-40, 0.1 percent bovine serumalbumin, 5×10⁻⁻⁵ M phenylmethylsulfonyl fluoride, and 0.017 TIU/ml ofapoprotinin and the resultant lysate was clarified by centrifugation at12,000×g. For immunoprecipitation reactions cell lysates were diluted3-fold with PBS and aliqouts (typically 180 μl) were mixed with 2-5 μlof antisera and incubated at 4° C. for 30 min. To immunoprecipitate thesecreted form of gD, 500 μl of conditioned medium was incubated with 2μl of antisera for 30 min at 4° C. Immune complexes were then adsorbedto fixed S. aureus cells by the method of Kessler (40a) and wereprecipitated by centrifugation at 12,000× g for 30 s. The S. aureuscells were then washed 3 times with wash buffer (PBS, 1 percent NP-40,0.3 percent sodium dodecyl sulfate), and the immune complexes wereeluted with 20 μl of polyacrylamide gel sample buffer (62.5 mM Tris-HClbuffer, pH 6.8 containing 10 percent glycerol, 5 percent2-mercaptoethanol, 0.01 percent bromophenol blue) at 90° C. for 3 min.After centrifugation for 30 s the supernatants were applied to 10percent polyacrylamide slab gels according to the method of Laemmli(45). A, immunoprecipitation of full length membrane bound gD from thegD12 cell line. B, immunoprecipitation of the cell associated form ofthe truncated gD from lysates of two independently derived cell lines (1and 2). C, immunoprecipitation of the truncated gD from the culturesupernatants of the two cell lines shown in B. (-), indicates controlrabbit antiserum; (+), indicates rabbit anti-HSV-1 antiserum (DakoCorp.).

As can be seen, evident are an intracellular form of 35,000 Daltons anda secreted and apparently glycosylated extracellular gD protein.

Preparation of Truncated gD Used for Immunization

gD10.2 cells were grown to confluence in polystyrene tissue cultureroller bottles (Corning 25140) in F12 medium supplemented with 7 percentcommercially dialyzed fetal calf serum, 50 μg/ml streptomycin, and 0.3μg glutamine. After reaching confluence the medium was removed and thecells were washed three times in the same medium lacking fetal calfserum and supplemented with 2 mg/ml Hepes buffer (serum free medium).The cells were then grown 3-4 days in serum free medium and theconditioned medium was then harvested and stored at -20° C. The mediumwas thawed at 37° C. and centrifuged at 5000 rpm for 20 min. in aSorvall GS-3 rotor. After centrifugation the pellet was discarded andthe supernatant was concentrated in an ultrafiltration apparatus(AMICON) YM-10 membrane equipped with a YM-5 ultrafiltration membrane.The resultant preparation was concentrated approximately 150-foldrelative to the starting material and contained approximately 8 mg ofprotein per liter. The preparation was then dialyzed extensively againstphosphate buffered saline (PBS) and used for immunization withoutfurther purification.

Immunization of Mice

Each 8-week old BALB/c mouse was immunized with 36 μg of proteincontained in 200 μl of an emulsion consisting of 50 percent aqueousantigen and 50 percent complete Freund's adjuvant. Each mouse wasimmunized at multiple intradermal and subcutaneous sites as follows: 25μl in each rear footpad, 50 μl in the tail, and 100 μl distributed among3-5 intradermal sites along the back. Four weeks after the primaryimmunization the mice were boosted with 36 μg of the protein as abovewith the exception that the emulsion was prepared with incompleteFreund's adjuvant. For the booster immunization each mouse received 200μl of the antigen emulsion distributed as follows: 50 μl in the tail,150 μl distributed among 5 intradermal sites along the back. 19 daysafter boosting approximately 500 μl of blood was collected from eachmouse by tail bleeding. The sera obtained from this bleed was used forin vitro neutralization studies (see below). 37 days after boosting themice were used for virus challenge studies. Control mice matched to theexperimentals with regard to age, sex and strain were immunized withhuman serum albumin (15 μg per mouse) using the same protocol as withthe experimentals.

In Vitro Neutralization

Sera from eleven mice immunized with concentrated gD10.2 culturesupernatant were tested for the ability to neutralize HSV-1 in vitro.Serially diluted mouse serum (2-fold dilutions: 1:8 to 1:16384) wereincubated with approximately 40 pfu of HSV-1 for 1 hr. at 37° C. inDulbecco's modified Eagle's medium (DMEM). After the serum incubation,each dilution was applied to approximately 40,000 Vero cells containedin each well of a 96 well tissue culture plate. After 3-4 days virusgrowth was determined by staining each well with 0.5 percent crystalviolet. Wells in which virus growth occurred showed no staining.Neutralization titers were calculated by determining the highest serumdilution which prevented virus induced cell death. All of the seratested (n=10) from mice immunized with gD10.2 supernatant materialshowed HSV-1 neutralization activity (range 1:16 to 1:512) and HSV-2neutralization activity (range 1:8 to 1:16). Control mouse sera (n=8)failed to provide any neutralization. Serum obtained from a mouseimmunized with HSV-1 gave a neutralizing titer of 1:32.

Virus Challenge

Eleven mice immunized with concentrated gD10.2 supernatant and 13control mice immunized with human serum albumin were challenged with10,000,000 pfu of HSV-1 (MacIntyre strain) by intraperitoneal injection.14 days after the injection of virus, none of the gD10.2 immunized miceshowed any indication of viral infection. In the control group, 7 of the13 mice were dead by day 14, 3 showed severe wasting and paralysis, and3 looked healthy. Statistical analysis (two tailed Fisher exact test)revealed that the difference between the immunized and control groupswas significant at the P=0.002 level. (See Table 1).

                  TABLE 1                                                         ______________________________________                                        No of            HSV1    HSV2   HSV1.sup.2,4                                                                         Challenge                              Expt.                                                                              Mice   Antigen  neut..sup.1                                                                         neut..sup.1                                                                          Paralyzed                                                                            Dead Alive                           ______________________________________                                        509C 11     gDtrunc.sup.3                                                                          1:16- 1:8-1:16                                                                             0      0    11                                                   1:512                                                    509D 13     HSA      0     0      3      7     3                              ______________________________________                                         .sup.1 Mouse sera were tested for HSV1 and HSV2 neutralization activity 1     days after the second secreted gD booster vaccination. Serially diluted       mouse sera (1:8-1:16384) were incubated with 40 forming units of HSV1 or      HSV2 for 1 hour at 37° C. Each dilution was applied to 40,000 Vero     cells contained in each well of 96 well microtitre wells. After 4 days,       the cells were stained with 0.5 percent crystal violet. Neutralization        titres were calculated by determining the highest serum dilution which        prevented virus growth.                                                       .sup.2 Mice were challenged by intraperitoneal injection of 1 ×         10.sup.7 plaque forming units of HSV1 (MacIntyre strain). Challenged mice     were observed for a period of three weeks for HSV1 infection.                 .sup.3 Each mouse was immunized with approximately 3 micrograms of            secreted gD in a 50 percent aqueous, 50 percent Freund's adjuvant             solution. Mice were immunized at multiple intradermal and subcutaneous        sites. Four weeks after the primary immunization, mice were boosted. Mice     were challenged 19 days after the booster immunization. Control mice were     immunized with an equivalent amount of human serum albumin (HSA).             .sup.4 Significant at p = 0.002 level.                                   

It was found that the truncated protein released into the medium fromgD10.2 cells was effective to protect mice from a lethal infection fromHSV-1.

Antigen Preparation for HSV-2 Virus Challenge

Amplified gD10.2.2 cells, grown in the presence of 250 nM methotrexate,were seeded into roller culture bottles (850 cm²) and were cultured inHam's F12 medium (GIBCO) supplemented with 7 percent fetal bovine serum.After the cells reached confluence (approximately 3 days), the culturemedium was removed, the cells were washed three times in phosphatebuffered saline (PBS) to remove serum proteins, and new "serum free"culture medium was added. The serum free medium consisted of Ham's F12medium containing 25 mM Hepes buffer. The cells were then cultured forthree days and the resultant conditioned medium was harvested and usedfor antigen preparation. Fresh serum-free medium was then added to thecells and the cycle of harvesting conditioned medium at three dayintervals was repeated an additional one or two times until the cellsdied or no longer adhered to the culture surface. gD10.2.2 conditionedserum-free medium was then filtered and centrifuged at low speed toremove cellular debris, and the resultant material was then concentratedten- to twenty- fold with an ultrafiltration device (YM-10 membrane,AMICON). The concentrated medium was then dialyzed overnight against PBS(3 changes of PBS, one liter per change). The resulting material wasthen assayed to determine the protein concentration and analyzed bypolyacrylamide gel electrophoresis to determine protein composition andto estimate the purity of the preparation. The material prepared by thisprocess was then used to immunize animals against HSV-2 infection asdescribed below.

Immunization of Mice Against HSV-2 Infection

Forty female BALB/c mice were obtained from the Charles RiverLaboratories (Boston, Mass.) and were immunized with the secreted gDprotein (gDtrunc) or human serum albumin (HSA) at 12 weeks of age. Forthe primary immunization against the secreted gD protein, the antigenwas adjusted to a concentration of approximately 70 ug per ml inphosphate buffered saline and was emulsified with an equal volume ofcomplete Freund's adjuvant. Each mouse was then immunized with 200 μl ofthis emulsion distributed as follows: 50 μl subcutaneously at a siteapproximately 1 cm from the base of the tail, 25 μl subcutaneously ineach rear footpad, and 100 μl distributed among 3-5 intradermal sitesalong the back. The mice were then boosted with the same antigen onemonth after the primary immunization. For the booster immunization theantigen was prepared by the same procedure as with the primaryimmunization with the exception that incomplete Freund's adjuvantreplaced complete Freund's adjuvant. For the booster immunization, 200μl of antigen emulsion was injected into each mouse and was distributedas follows: 50 μl in the tail, 25 μl subcutaneously in the loose skinabove each thigh, and 100 μl distributed among 3-5 intradermal sitesalong the back. The control group of mice was immunized according to thesame protocol as the experimental group of mice with the exception thathuman serum albumin replaced the secreted gD protein as the immunogen.Serum was collected from the mice 24 days after boosting for use in invitro neutralization studies.

HSV-2 Virus Challenge

Both experimental (secreted gD injected) and control (HSA injected)groups of mice were challenged by an intraperitoneal injection of HSV-2(MS strain) 31 days after the booster immunization. Each mouse received2×10⁵ pfu of virus in 100 μl of Dulbecco's modified Eagle's medium(DMEM) containing 10 percent fetal bovine serum. LD 50 experimentsrevealed that this amount of virus represented 100-500 times the amountof virus required to kill 50 percent of a population of normal(uninjected) BALB/c mice. The virus injected mice were observed for aperiod of 3 weeks. All of the control mice (HSA injected) died within 9days of the virus challenge. All of the mice vaccinated with thesecreted gD protein survived the full three weeks and appeared normal(i.e., they did not exhibit wasting or paralysis).

                  TABLE 2                                                         ______________________________________                                        No of            HSV1    HSV2   HSV1.sup.2                                                                           Challenge                              Expt.                                                                              Mice   Antigen  neut..sup.1                                                                         neut..sup.1                                                                          Paralyzed                                                                            Dead Alive                           ______________________________________                                        579C 15     gDtrunc  1:1024-                                                                             1:512- 0       0   15                                                   1:2048                                                                              1:1024                                             579D 25     HSA      0     0      0      25    0                              ______________________________________                                         1. Mouse sera were tested for HSV1 and HSV2 neutralization activity 19        days after the second secreted gD booster vaccination. Serially diluted       mouse sera (1:8-1:16384) were incubated with 40 forming units of HSV1 or      HSV2 for 1 hour at 37° C. Each dilution was applied to 40,000 Vero     cells contained in each well of 96 well microtitre wells. After 4 days,       the cells were stained with 0.5 percent crystal violet. Neutralization        titres were calculated by determining the highest serum dilution which        prevented virus growth. Values indicated represent the average                neutralization titers.                                                        2. See text above for the details of the HSV2 challenge.                 

The advantages of using the truncated protein for diagnostic and vaccineapplications is that, being secreted into the extracellular medium, itis contaminated with far fewer proteins than would be found in awhole-cell preparation.

It will be noted that the present invention uses a permanent cell lineto produce the protein. Upon transfection the vector is incorporatedinto the genome of the cell line and can produce the protein withoutcell lysis. The cell line can thus be used for continuous production ofthe protein, especially in the truncated form which is secreted from thecell. For example, the cells expressing truncated protein can becontinuously used in a perfusion system by constantly removingantigen-rich medium from the cells and replacing it with fresh medium.

The particular cell line used here was a CHO line deficient in dhfrproduction, transfected with a vector containing a dhfr marker. Byexposing the cell line to methotrexate (Mtx) under suitable conditions(54) the dhfr production and hence the linked gD protein production canbe amplified. Three cell lines derived by transfection of the truncatedgD gene into dhfr⁻ CHO cells were plated in parallel, labeled with ³⁵S-methionine, and immunoprecipitated as described in FIG. 2. Lanes 1 and2 indicate the amount of secreted gD immunoprecipitated from 500 μl ofculture medium conditioned by two independently isolated cell linesbefore selection with methotrexate. Lane 3 indicates the amount oftruncated gD imunoprecipitated from an equal volume of culture mediumfrom a cell line (gD10.2.2) selected for growth in 250 nM methotrexate.Rabbit anti-HSV-1 antibodies (Dako Corp.) were used for theimmunoprecipitations shown in lanes 1-3. Lane 4 represents a controlimmunoprecipitation of 500 μl of medium conditioned by the gD10.2.2 cellline with normal rabbit serum.

To quantitate the relative amounts of truncated gD secreted into theculture medium by cell lines before and after selection in methotrexate,a competitive ELISA assay was performed. gD12 cells expressing amembrane-bound form of gD were plated out and fixed with glutaraldehydeto the surface of 96 well microtiter plates as previously described.Conditioned medium from various cell lines known to produce thetruncated gD was serially diluted across the microtiter plate and wasincubated with a fixed quantity (2 μl) of rabbit anti-HSV-1 antibody(Dako Corp) for 1 hr at 20° C. Unbound antibody and soluble truncatedgD-antibody complexes were removed by washing each well 3 times withPBS. Horseradish peroxidase coupled to goat anti-rabbit IgG was thenreacted with the fixed cells for 1 hr at 20° C. and unbound antibody wasremoved by washing 3 times with PBS. The colorometric substrate, OPD(o-phenylene diamine), was then added to each well and allowed to reactwith the bound horseradish peroxidase-antibody complexes for 15 min. Thereaction was terminated by the addition of sulfuric acid to a finalconcentration of 0.25N. The absorbance of the OPD in each well wasdetermined with the use of an automated microtiter plate scanner(Titertek multiskan) and dilution curves were plotted. The binding ofanti-HSV-1 antibodies to the parental CHO cell line was used to measurethe extent of nonspecific binding at each dilution. The amount oftruncated gD in each culture supernatant was inversely proportional tothe amount of absorbance in each well. Open circle, binding ofanti-HSV-1 antibodies to gD12 cells in the presence of mediumconditioned by cells secreting truncated gD before amplification withmethotrexate. Closed circle, binding of anti-HSV-1 antibodies to gD12cells in the presence of medium from gD10.2.2 cells selected for growthin 250 nM methotrexate. Open square, binding of anti-HSV-1 antibodies togD12 cells in the presence of 100-fold concentrated medium fromunamplified cells secreting truncated gD. This procedure was carried outon the gD10.2 cell line to produce an amplified cell line gD10.2.2 whichwas capable of growth in 250 nM Mtx and which secreted approximately20-fold more truncated gD into the culture medium than the parentalgD10.2 cell line (see FIGS. 10 and 11).

The dhfr marker/amplification system can be used with other cells whichare able to acquire and stably incorporate foreign DNA.

The success of this invention in demonstrating that a truncated form ofa membrane bound protein, lacking that part of thehydrophobic-hydrophilic carboxy-terminal region responsible for bindingit to the membrane, can yet be immunogenic indicates that similarresults can be expected with other immunogenic membrane bound proteins,thus providing an improved source of vaccine against viruses, parasitesand other pathogenic organisms.

In the foregoing example, the DNA of gD protein was truncated at residue300 because there was a convenient restriction site there. This had theresult that the carboxy-terminal hydrophobic/hydrophilic region wascompletely removed,-as can be seen from the hydropathy plot of FIG. 2;indeed an additional preceding region was removed from residue 301 to332 without, apparently, destroying the immunogenic character of theprotein. It would seem to follow, therefore, that with this protein, andprobably with other immunogenic membrane bound proteins, the extent oftruncation could be considerably less if desired, so long as it has theeffect of removing the membrane binding character so that the protein issecreted into the surrounding medium.

Example 2

Example 2 relates to an HSV-2 gC protein (formerly designated a gFprotein).

Cells, Virus, and DNA Isolation

HSV-2 (strain G) was grown on HEp 2 cells after infecting the cellculture at an input multiplicity of 0.1 for 3 days at 33° C. inDulbecco's Modified Eagles Medium containing 10 percent fetal bovineserum and antibiotics. HSV-2 DNA was isolated by proteinase K digestionfollowed by CsCl ultracentrifugation as described (23).

DNA Manipulations

Restriction enzymes, DNA polymerase Klenow fragment, T4 DNA ligase, andT4 polynucleotide kinase were purchased from Bethesda Research Labs andwere used according to the suppliers' directions.

Molecular Cloning of HSV-2 DNA Restriction Fragments

The EcoRI "P" fragment, which corresponds to approximate map position˜0.650 of the HSV-2 genome, was isolated from EcoRI digested HSV-2 DNAon 5 percent acrylamide gels. The isolated fragment was cloned into EcoRI digested pUC9 (28). This plasmid was called pUC-RlP.

The pUC-RlP subclone was then used to localize a Sac I fragment of theHSV-2 genome which contained the Eco RI "P" fragment. Southern blotexperiments (27) revealed that a 4.9 kb Sac I fragment of HSV-2contained the EcoR1 "P" fragment. This fragment was isolated on 0.7percent agarose gels and was cloned into a pBR322-derived plasmid whichcontained a unique Sac I site (55). This plasmid was called pBRSac1-"E".Further restriction enzyme analysis of pBRSac1-"E" demonstrated a 2.9 kbSal I fragment with sequences homologous to the Eco RI "P" fragmentwhich was subcloned into Sal I digested pUC9 as described above. Thisplasmid was called pgC₂ Sal2.9.

DNA Sequence Analysis of Cloned HSV-2 DNA

The majority of DNA sequences were determined using the dideoxynucleotide chain termination technique. Various fragments were subclonedinto the replicative form of the ml3 phage vectors mp7, mp8, and mp9,and the DNA sequence was determined as described previously (29). Insome cases, fragments were ³² P-labelled at their 5' ends with γ³² P-ATPand T4 polynucleotide kinase and the DNA sequence of the fragment wasdetermined by using the chemical degradation method (56).Computer-assisted analysis of DNA and protein sequence data wasperformed using the HOM program (57). The hydropathy of the deducedamino acid sequences was analyzed using a width of 12 amino acids and ajump of 1 (31a).

Southern Blot Analysis of HSV-2 DNA

Restriction endonuclease digested HSV-2 DNA and plasmid DNA werefractionated on 1.5 percent agarose gels and blotted onto nitrocelluloseusing standard procedures. The single-stranded ends of the Sac IIfragment, marked with a star in FIG. 12, were filled in with the Klenowfragment of DNA polymerase 1, and the resultant blunt-ended fragment wasligated to Sma I digested ml3mp7 replicative form (29) with T4 DNAligase. The single-stranded DNA prepared from this ligation andtransfection was used as a template for the synthesis of ³² P-labeledsingle-stranded probe DNA of high specific activity (1×10⁹ cpm/μg) usingthe Klenow fragment of DNA polymerase I. Hybridizations were performedusing standard procedures (27,58).

RESULTS

Molecular Cloning of the gF Coding Region of the HSV-2 Genome

The strategy adopted for the isolation of the gF gene of HSV-2 was basedon the assumption that this gene was colinear with the HSV-1 gC gene.This assumption was supported by the recent finding that a 75,000 daltonglycoprotein, gF, with antigenic relatedness to HSV-1 glycoprotein C isfound in HSV-2 and that the gene for this protein is approximatelycolinear with the HSV-1 gC gene (22d,59). In addition, the isolation ofa monoclonal antibody which binds to both HSV-1 gC and HSV-2 gF furthersuggested that these two proteins may be homologous to each other (22f).It was thus reasoned that DNA sequence analysis of the HSV-2 genomicregion which is colinear with the HSV-1 gC gene would result in thederivation of protein sequence information which would localize theHSV-2 gF gene.

The 600 basepair Eco RI "P" fragment of the HSV-2 genome has been shownto map at position ˜0.650 (12). This region is approximately colinearwith the known coding region of the HSV-1 gC gene which maps betweenapproximately 0.630 and 0.640 of the HSV-1 genome (59). This fragmentwas isolated from an Eco RI digest of HSV-2 DNA, cloned in the plasmidpUC9 (28), and its DNA sequence was determined (29,56). Comparison ofthe resultant sequence with the HSV-1 gC sequence (59) revealed aremarkable degree of sequence homology between the Eco RI "P" fragmentand the 3' end of the HSV-1 gC coding region. Thus, the Eco RI "P"fragment was subsequently used as a probe to isolate a Sac I restrictionendonuclease fragment from HSV-2 genomic DNA that overlapped the Eco RI"P" fragment sufficiently to include the remainder of the HSV-2 genewhich was homologous to the HSV-1 gC gene. FIG. 12 illustrates the stepstaken to isolate a 2.9 kb SalI fragment from the HSV-2 genome whichcontained the Eco RI "P" fragment and which was used for subsequent DNAsequence analysis.

DNA Sequence Analysis of the Eco RI "P" region of the HSV-2 Genome

The 4.3 kb Sac I "E" fragment, which was isolated from the HSV-2 genomebased upon its sequence homology to the Eco RI "P" fragment, was furtherdigested to give a 2.9 kb Sal I fragment which was termed pgC₂ Sal2.9.FIG. 12 illustrates the fragments from pgC₂ Sal2.9 which were subjectedto DNA sequence analysis using either the dideoxy-nucleotide sequencingprocedure (29) or the chemical degradation procedure (56). In addition,this figure shows the position of the Eco RI "P" fragment within pgC₂Sal2.9 as well as the position of a BgI II site which corresponds to theright hand end of the BgI II "N" fragment at position ˜0.628 of theHSV-2 genome (12).

Specifically, FIG. 12 shows the cloning of pgC₂ Sal2.9, the HSV-2 regionwhich maps colinearly with HSV-1 gC. The region of the HSV-2 genomemapping from ˜0.61-0.66 was cloned as a Sac I fragment (pBRSac "E")using the 600 basepair "P" fragment as a probe. A Sal I subclone ofpBRSac "E", pgC₂ sal2.9, was used for DNA sequence analysis. Arrowsrefer to the sequenced regions , and the location of a major 479 aminoacid open reading frame derived from the sequence is illustrated.Various restriction sites are illustrated, including the Eco RI siteswhich delineate the Eco RI "P" fragment, and the Bgl II site which isfound at the right end of the Bgl II "N" fragment (map position ˜0.628)(26). The Sac II fragment marked with a star (*) was used in Southernblotting experiments to investigate the deletion which appears in thisregion (see results). Other sites were used for DNA sequencingexperiments. Sm; Smal, Sa; Sac II, Rs: RsaI, Bg; Bgl II, Pv; Pvu II, R1;Eco RI.

FIG. 13 illustrates the DNA sequence obtained from pgC₂ Sal2.9 comparedwith the DNA sequence of the HSV-1 ₉ C region (59). The HSV-1 gC region(HSV-1) and the sequence obtained from pgC₂ Sal2.9 (HSV-2) were comparedusing the HOM program (57). Because various deletions were utilized tomaximize sequence overlap, all positions, including spaces, have beennumbered for clarity. Stars are placed over non-matching nucleotides.The underlined "A" residue at position 43 of the HSV-1 sequence is theapproximate transcriptional start site of the gC mRNA (59). "TATA" 1 and"TATA" 2 are the probable transcriptional control regions for the HSV-1gC mRNA and the 730 base mRNA, respectively (59,60). The inserted Tresidue at position 1728 of the HSV-1 sequence was discovered byresequencing this region (M. Jackson, unpublished) and was found tointroduce an in-phase stop codon at positions 1735-1737 which washomologous to the stop codon for the HSV-2 major open reading frame. Theposition of the 730 base mRNA initiation codon of HSV-1 is shown atposition 2032-2034, as is the position of a second HSV-2 initiationcodon at position 1975-1977.

Referring again to FIG. 13, the illustrated derived sequence of HSV-2was compared with the DNA sequence of the gC gene region of HSV-1 (59)which showed an overall sequence homology between these two fragmentswas approximately 68 percent. However, certain regions of the sequenceshowed either a much higher or lower degree of sequence homology thanothers. For example, the sequences between positions 0 and 570 of theHSV-1 and HSV-2 sequences showed only 51 percent homology, while theregion between position 570 and 1740 showed a much higher degree ofsequence homology (80 percent). An additional highly homologous region(70 percent) was also found at the end of the two sequences fromposition 1975 to position 2419. In addition to the nucleotide sequencechanges, the two genomes showed various deletions or insertions whencompared to each other. The most notable was an 81 basepair region foundat position 346-426 of the HSV-1 gC sequence which is missing from theHSV-2 genome. From this overall sequence comparison it appeared thatthere was a high degree of sequence homology between the HSV-1 gC regionand the HSV-2 region sequenced here.

Frink et al. (59) have found that the 5' end of the 2,520 base mRNAencoding HSV-1 gC maps to the underlined A residue at position 43 ofFIG. 13. In addition, they pointed out an AT-rich "TATA" box (60)sequence approximately 22 basepairs 5' to this residue. Comparison-ofthe two sequences shown in FIG. 13 shows that the HSV-1 and HSV-2sequences both contained the identical sequence, CGGGTATAAA, in thisregion. This sequence is identical to that reported previously byWhitton et al. (61), which is found to occur at the "TATA" box regionsin many of the HSV-1 and HSV-2 sequences determined thus far. Thisconserved sequence is also followed by a G-rich region in both virusgenomes. In addition to this putative transcriptional-control region, asecond "TATA" box was found in both sequences at position 1845-1849 ofFIG. 13. This second "TATA" box has been hypothesized to control thetranscription of a 730 base mRNA in the HSV-1 genome (59). Both HSV-1and HSV-2 contain this sequence surrounded by GC-rich flankingsequences, including a CGGGCG sequence which is similar to the CGGGsequence preceding the first "TATA" box. In addition, both genomesencode open reading frames 3' to these second "TATA" boxes, which willbe discussed below.

In order to determine if the 81 basepair deletion described above wasactually found in the HSV-2 genome or if it was an artifact of cloningor sequencing, Southern blot analysis of the HSV-2 genomic DNA and thecloned HSV-2 DNA was performed. A ³² P-labeled probe was prepared from aSac II fragment (see fragment in FIG. 12) which spans the region missingthe 81 nucleotides. If the HSV-2 genomic DNA is missing the 81 basepairregion, then a Sma I-BglII fragment spanning this region will be 576basepairs, a Sma I fragment will be 662 basepairs, and a Sac II fragmentwill be 195 basepairs.

FIG. 14 illustrates Southern blot analysis of HSV-2 genomic DNA and pgC₂Sal2.9 DNA. The region spanning the 81 basepair region missing in theHSV-2 sequence shown in FIG. 13 (HSV-2 positions 346-426) was analyzedusing the Sac II fragment marked with a star in FIG. 12 which overlapsthe deleted region. Lanes 1-3 are restriction digests of HSV-2 genomicDNA, and lanes 4-6 are restriction enzyme digests of pgC₂ Sal2.9. Thedigested DNAs were electrophoresed on 1.5 percent agarose gels,denatured, blotted onto nitrocellulose, and probed with the ³² P-labeledSac II fragment. (The arrow shows the position of the 564 base pair HindIII fragment of phage λ DNA.) Lanes 1,6; Sma I+Bgl II: lanes 2,5; Sma1:Lanes 3,4; Sac II.

The results shown in FIG. 14 demonstrate that the predicted restrictionsites surrounded the region missing the 81 basepairs in both the HSV-2genomic DNA and the cloned HSV-2 DNA. In addition, the HSV-2 genomicfragments and the cloned fragments comigrated exactly, demonstratingthat the deletion is not an artifact of cloning or sequencing.

Analysis of the Major Open Reading Frame Within the HSV-2 2.9 kb Sal IFragment

Analysis of the potential coding sequences within the 2.9 kb Sal I DNAfragment of HSV-2 revealed an open reading frame of 479 amino acidswhich began with the methionine encoded at position 199-201 of the HSV-2sequence shown in FIG. 13 and ended at the TAA termination codon atposition 1735-1737 of the HSV-2 sequence in this figure. As can be seenfrom FIG. 13, both the HSV-1 gC protein and the HSV-2 open reading frameinitiate at approximately the same position in the two sequences,relative to the "TATA" box homologies. In addition, while it initiallyappeared that the HSV-2 open reading frame found in this regionterminated 12 codons before the HSV-1 gC gene, resequencing of thecarboxy-terminal region of the gC gene sequence (M. Jackson,unpublished) of HSV-1 strain F revealed that the sequence reported byFrink et al. (59) was missing a thymidine nucleotide after position 1727and that insertion of this residue resulted in a translated HSV-1 gCprotein terminating at the same place as the HSV-2 open reading frame(1735-1737 of FIG. 13). Thus, when taking the various deletions andinsertions into account, as illustrated in FIG. 13, the HSV-1 gC geneand the HSV-2 open reading frame show a very high degree of overlap.

FIG. 15 illustrates translation of the HSV-2 large open reading frameand comparison with the HSV-1 gC amino acid sequence. The single letteramino acid symbols were used. HSV-1-gC refers to the HSV-1 gC sequence,and HSV-2 gF refers to the HSV-2 open reading frame sequence. Theproteins were compared using the HOM program, which maximized homologiesby inserting gaps where necessary (57). Stars are placed overnon-homologous amino acids. Putative N-linked glycosylation sites (NXSor NXT) (62) are shaded, and cysteine residues (C) are boxed. Only aminoacids, and not spaces are numbered. 15B illustrates translation of thesecond HSV-2 open reading frame and comparison with the HSV-1 730 basemRNA protein. 730 ORF HSV-2 is the incomplete amino acid sequence of thesecond HSV-2 open reading frame from positions 1975-2406 of the HSV-2sequence shown in FIG. 13. 730 ORF HSV-1 is the amino acid sequencederived for the protein encoded by the 730 base mRNA of HSV-1 (59).Conserved amino acid changes, with respect to charge, are marked (C) andnonconserved changes, with respect to charge, are marked (N) in bothFIG. 4A and 4B.

FIG. 15 illustrates the high degree of sequence homology between theHSV-1 gC gene and the 479 amino acid HSV-2 open reading frame. The first19 amino acids contain approximately 80 percent sequence homology withthe changes in the first 25 amino acids being all conservative withrespect to charge. From residue 124 of HSV-1 gC (residue 90 of the HSV-2sequence) to the end of both proteins there is about 74 percent sequencehomology with 75 percent of the amino acid changes being conservativewith respect to charge. Five putative N-linked glycosylation sites (NXSor NXT (62)) are conserved between the two proteins, and all 7 cysteineresidues are located in homologous positions relative to the C-terminus.In addition to the overall conservation of sequences in thecarboxy-terminal three-fourths of the proteins, there are also largeregions of contiguous amino acid sequence homology up to 20 residues inlength (i.e., position 385-405 of the HSV-1 sequence and 352-372 of theHSV-2 sequence). It may be concluded from this sequence comparison thatthe open reading frame in this region of the HSV-2 genome encodes aprotein which is homologous to HSV-1 gC.

While the HSV-2 protein encoded in this region shows a remarkable degreeof sequence homology to the HSV-1 gC sequence, there are several notabledifferences between the two sequences. The most striking difference is adeletion of 27 amino acids in the HSV-2 sequence which are found in theHSV-1 gC sequence from residues 50-76 (FIG. 15) and which corresponds tothe 81 basepair deletion described above. In addition to this largedeletion, both sequences show minor deletions of one or two amino acids.All of these deletions are found in the amino-terminal regions of theproteins. In addition to these deletions, there are a large number ofamino acid changes in the amino-terminal region of the proteins whichare clustered between residues 29-123 of the HSV-1 gC sequence (residues31-90 of the HSV-2 sequence). Only 30 percent of the amino acids in thisregion are homologous, with much of this homology due to conservedproline residues. 43 percent of the amino acid substitutions found inthis region are non-conservative with respect to charge. The only otherregions which showed such a large number of changes are acarboxy-terminal hydrophobic domain (residues 476-496 of the HSV-1sequence and 443-463 of the HSV-2 sequence) where the proteins are 55percent homologous but where all the changes are conserved, uncharged,hydrophobic amino acids and the carboxy-termini of the proteins wherethe sequences are only 25 percent homologous, but where the overallamino acid composition is similar (residues 500-512 of the HSV-1sequence and 467-479 of the HSV-2 sequence). While five of the putativeN-linked glycosylation sites are conserved between the two proteins, theHSV-1 gC sequence contains two more sites than the HSV-2 sequence (9versus 7 total). The HSV-1 gC sequence contains 2 N-linked glycosylationsites in the 27 amino acids deleted from the HSV-2 sequence, and anoverlapping pair of sites between residues 109 and 112 of FIG. 15. TheHSV-2 sequence contains two N-linked glycosylation sites not found inthe HSV-1 sequence, one of which is proximal to the amino terminus.

In order to more fully examine the possible structural homologiesbetween the HSV-1 and HSV-2 sequences, hydropathy analysis was performed(31a). FIG. 6 illustrates hydropathy analysis of the HSV-1 gC proteinand the HSV-2 major open reading frame protein. The hydropathy of eachprotein was determined using the program of Hopp and Woods (31a).Hydrophobic regions are above the midline and hydrophilic regions arebelow the midline. Stretches of 12 amino acids were analyzed, and theaverage hydropathy was calculated. Putative asparagine-linkedglycosylation sites (62) are marked (0). gC-1: HSV-1 gC proteinhydropathy. gC-2 (gF): HSV-2 major open reading frame proteinhydropathy.

FIG. 16 shows that both proteins exhibited an extraordinary degree ofstructural homology based on the hydrophilic and hydrophobic propertiesof the amino acid sequences. Each show an N-terminal hydrophobic domainfollowed by a stretch of hydrophilic amino acids which contain either 6of 9 total (HSV-1) or 3 of 7 total (HSV-2) putative N-linkedglycosylation sites. The peaks and valleys which follow this hydrophilicregion are very similar in both proteins, including the hydrophilicdomain containing the final N-linked glycosylation site. Thecarboxy-termini of both proteins shows a very hydrophobic 20 residueregion followed by a hydrophilic carboxy-terminus. The 27 contiguousamino acids found exclusively in the HSV-1 gC protein appear to encode arelatively hydrophilic region between residues 50-76 (FIG. 16). Inconclusion, this analysis reveals that the hydropathic features of boththe HSV-1 gC and the HSV-2 protein are very similar and that the leastconserved amino-terminal regions of the proteins are found inhydrophilic regions which have the potential to be highly glycosylated.

Analysis of the Second HSV-2 Open Reading Frame

Translation of the final 431 basepairs of the HSV-2 sequence shown inFIG. 2 (residues 1975-2406) revealed a second open reading frame of 105amino acids. Although the sequence information reported here isinsufficient to contain the entire HSV-2 second open reading frame,comparison of this sequence with the open reading frame encoded by the730 base mRNA of HSV-1 reported by Frink et al. (10) also revealed ahigh degree of sequence homology. As can be seen in FIG. 4b, the twosequences showed 75 percent sequence homology in the overlappingregions, with about 90 percent of the amino acid changes beingconservative with respect to charge. The major difference between thetwo sequences was a 19 amino acid N-terminal region which was found inthe HSV-2, but not HSV-1 sequence. Thus, although the function of theprotein encoded in this region is unknown, the proteins from HSV-1 andHSV-2 show a considerable degree of sequence homology.

DISCUSSION

The above results demonstrate that the HSV-2 genome encodes a colinearlymapping homologue of the HSV-1 glycoprotein C. The colinearity of thesequences found here is strengthened by the finding of a sequence 3' ofthe HSV-2 major open reading frame which apparently encodes a homologueof the HSV-1 730 base pair mRNA (10). Previous mapping of the HSV-2 gFgene (33), together with the properties described here for the majoropen reading frame in this region of the HSV-2 genome including severalpotential N-linked glycosylation sites and an apparent amino-terminalsignal sequence (5) as well as a putative carboxy-terminal transmembranedomain (28) allow the conclusion that the HSV-2 protein described hereis the glycoprotein, gF. In addition, the size of the translated HSV-2protein (˜52,000 daltons) is similar to that reported for theendoglycosidase H-treated, native size for HSV-2 gF (54,000 daltons)(22d). Finally, the large extent of amino acid sequence homology as wellas the conservation of several potential N-linked glycosylation sitesand of all 7 cysteine residues indicates structural homology betweenHSV-1 gC and HSV-2 gF. These results, then, strongly suggest that theHSV-1 gC protein and the HSV-2 gF protein are homologous to each other.

These results help explain previous results which demonstrated that theHSV-2 gF and HSV-1 gC proteins were mainly type-specific, but that theydid have type-common determinants (17,22d,22f,43). Since severalprevious studies (17,18,43) demonstrated that these proteins inducedpredominantly type-specific antibodies, it is reasonable that the mostantigenic regions of the proteins are found within the more divergentN-terminal sequences which follow the putative hydrophobic signalsequences. The hydrophilic nature of the divergent regions, along withtheir high content of potential N-linked glycosylation sites (62),suggests that these regions would be located on the surface of theprotein. Exposure of these divergent sequences to the outside of theproteins may be responsible for the generation of type-specificantibodies directed against these divergent epitopes. However,type-common antibodies could likely also be generated by the more highlyconserved carboxy-terminal three-fourths of the proteins, sincehydrophilic regions conserved between gC and gF could be exposed to theoutside of the proteins and may be, in one case, glycosylated (residues363-366 of HSV-1 gC and 330-332 of HSV-2 gF). Thus, HSV-1 gC and HSV-2gF share both type-specific and type-common determinants, but it appearsthat the type-specific determinants are more antigenic.

Although an explanation of the type-specific and type-commondeterminants of gC and gF is not known, it is possible that the proteinshave at least two functions, one of which is important for the viabilityof both viruses, the type common domain, and one of which is specificfor each virus type, the type-specific domain. While the function(s) ofgC and gF is at present unknown, and while viable gC minus mutants ofHSV-1 have been isolated in vitro (65), it is not clear that either gCor gF are indispensable to the viruses during in vivo infection of thehuman host and the establishment of latency. It is possible that atleast some of the biological differences between HSV-1 and HSV-2,including predilection for site of infection and virulence, may be dueto the marked structural differences between the amino-terminal regionsof gC and gF. It may be concluded, even in the absence of any functionalknowledge of these proteins, that different-selective pressures must beoperating on the divergent and conserved domains of gC and gF.

Previous sequence comparison of the gD genes of HSV-1 and HSV-2 (58)demonstrated that the amino-terminal signal sequence (63) and thecarboxy-terminal transmembrane domain (64) were able to tolerate a largenumber of mutations as long as the substituted amino acids werehydrophobic. The gC and gF sequence comparison demonstrates a similarfinding in the carboxy-terminal, putative transmembrane domain (64) fromresidues 476-496 of gC and 443-463 of gF. The large number ofheterologous hydrophobic substitutions in this region suggests that, asin gD, any amino acid which is lipid-soluble can be tolerated in thisregion. In contrast to gD, however, the amino-terminal signal sequencesof gC and gF are highly homologous in the first 19 residues. Thus,either this region has an important conserved function other thandirection of the glycoproteins into the rough endoplasmic reticulum (5),or there may be an overlapping gene or other functional sequence in thisregion of the genome which must be conserved (66).

Although insufficient HSV-2 sequence is presented here for a completecomparison, the region 5' to the start of HSV-1 gC mRNA transcriptionshows an identical CGGGTATAA sequence in both the HSV-1 and HSV-2genomes. In addition, both sequences are followed by a G-rich regionimmediately preceding the start of transcription. Thus, as waspreviously found for the gD genes of HSV-1 and HSV-2, upstream sequencehomologies exist between the two virus types which suggest thepossibility that these regions are involved in transcriptionalregulation of these genes. Interestingly, the second "TATA" box homologyfound in both virus genomes, which probably controls transcription ofthe 730 base mRNA (59,60), also shows a relatively high degree ofsequence homology in HSV-1 and HSV-2. These "TATA" boxes are preceded byCG-rich sequences, which are similar, but not identical, to thosepreceding the first "TATA" regions shown in FIG. 13, and they are bothfollowed by a 14 basepair region showing ˜80 percent sequence homology.The entire region of homology surrounding this region is only 33basepairs with an overall sequence homology of ˜75 percent. If thisregion is involved in transcriptional regulation of the 730 base mRNA,then it appears that a relatively short sequence may be sufficient forrecognition by transcriptional regulatory elements.

The results demonstrate that the HSV-1 gC and HSV-2 gF glycoproteins arehighly homologous, and that they encode type-common and type-specificdomains. Since the two proteins do show significant sequence homology,and since they apparently map colinearly, we favor the proposal ofZezulak and Spear (22d) to rename HSV-2 gF as HSV-2 gC or gC-2. Inaddition, the sequencing data reported here opens the way for afunctional analysis of the gC-1 and gC-2 proteins by the interchange ofvarious type-specific regions between the two proteins in vitro andexpression of the chimaeric sequences in mammalian cells (67) or byreincorporation of these regions back into the virus (68).

It is believed that the cloned gC-2 glycoproteins may be expressed andformed into a vaccine in a manner analogous to that set forth in Example1.

It is further believed that a vaccine which includes a mixture of suchrecombinant gC and gD glycoproteins would be significantly moreeffective as a vaccine against HSV-1 and HSV-2 than one based uponeither glycoprotein alone.

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What is claimed is:
 1. A vaccine effective against herpes simplex virustype 1 or type 2 pathogen comprising a membrane-bound polypeptide havingexposed antigenic determinants that raise neutralizing antibodiesagainst in vivo challenge by said pathogen, said polypeptide beingfunctionally associated with a membrane of a recombinant, stable,mammalian continuous cell line used in its production, said polypeptidecomprising a gD glycoprotein of herpes simplex virus type 1 or type 2.2. A vaccine effective against herpes simplex virus type 1 or type 2pathogen comprising a membrane-free polypeptide having exposed antigenicdeterminants that raise neutralizing antibodies against in vivochallenge by said pathogen, said polypeptide produced in a recombinant,stable, mammalian continuous cell line, said polypeptide comprising afull-length or truncated gD glycoprotein of herpes simplex virus type 1or type
 2. 3. A vaccine effective against herpes simplex virus type 1 ortype 2 pathogen comprising a truncated, membrane-free polypeptideaccording to claim 2, said polypeptide devoid of membrane-bindingdomain.
 4. A vaccine according to claim 3 wherein said truncated,membrane-free golypeptide comprises the N-terminal region of gDpolypeptide up to about amino acid residue
 300. 5. A method of producinga vaccine according to claim 3, wherein DNA encoding said truncatedmembrane-free polypeptide is prepared lacking sequence encoding themembrane-binding domain, comprising incorporating the prepared DNA intoan operative expression vector, stably transfecting a mammaliancontinuous host cell line with said vector, collecting the polypeptideas a secretion product, and incorporating said polypeptide into vaccineform.
 6. A method according to claim 5 or 22 wherein the cell line is aCHO cell line deficient in the production of dhfr and the vectorcontains a dhfr selectible marker.
 7. A method of producing a vaccineaccording to claim 2 or 3, said method comprising stably transfecting amammalian continuous host cell line with an expression vectoroperatively encoding said polypeptide, collecting said polypeptide as asecretion product and incorporating said polypeptide into vaccine form.8. A method according to claim 7 wherein the cell line is a CHO cellline deficient in the production of dhfr and the vector contains a dhfrselectible marker.
 9. A method of making a vaccine useful against invivo challenge with herpes simplex virus type 1 or type 2, comprisingincorporating a truncated gD polypeptide from herpes simplex virus type1 or type 2 into vaccine form.
 10. A vaccine according to claim 1, 2 or3 wherein said polypeptide solely consists of gD glycoprotein of herpessimplex virus type 1 or type 2.